Heterocyclic FRETdye cassettes for labeling biological molecules and their use in DNA sequencing

ABSTRACT

Exploitation of suitably functionalized heterocyclic molecules, in the design and synthesis of Fluorescence Resonance Energy Transfer (FRET) cassettes and their corresponding dideoxynucleotide terminators culminated into efficient reagents for DNA sequencing. Additionally, these FRET cassettes/terminators, of the present invention, derived from different classes of heterocyclic systems have high potential to be used for general labelling of biological molecules to generate highly sensitized signals. Their preparation, energy transfer efficiency, and use as labels, specifically, in DNA sequencing reactions is disclosed.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention relates to novel heterocyclic FRET (fluorescenceresonance energy transfer) cassettes, which can be used to label nucleicacids and proteins for a wide variety of molecular biology and DNAsequencing applications.

The currently accepted practice of high throughput gene sequencingemploys, as a general rule, four differently labeled Energy Transfer(ET) dye terminators based on the Forster Resonance Energy Transfer(FRET) mechanism to read out the sequence by exciting at one excitationwavelength of the donor and measuring the emissions at the wavelength ofthe four acceptors conjugated to the four individual nucleic acid bases.

However, the currently available ET terminator sets suffer from lowbrightness. This low brightness is due to the inefficiencies in thetransfer of energy absorbed by the donor to the acceptors andre-emitance at the emission wavelength of the acceptor. Thisinefficiency arises because of the structural linkages used to join thedonor and the acceptors as well as the bases together to form the dyeterminators are less than optimal.

The FRET efficiency mainly depends upon the relativedipole-induced-dipole orientation of the participating dyes. Thefunctional groups' orientations with which these dyes are covalentlybonded onto the core linker molecule would determine the relativedipole-dipole orientations of the dyes. Heterocyclic and alicyclicmolecules with suitable functional groups for making covalent bonds withfluorescent dyes and a molecule of biological interest would serve ascassette cores by virtue of orienting the functional groups in 3D,thereby defining fixed positions for the attached groups. In order toderive highly efficient FRET dye cassettes and turn them into highlysensitive DNA sequencing terminators, heterocyclic systems withdifferent structures and ring sizes were chosen to serve as the corecassette molecules.

In this invention, we offer a novel set of dye labeled cassettes and thecorresponding terminators which is brighter than the currently availableterminators. The increase in brightness for the set of dye terminatorsof this invention and the corresponding improvement in signal to noiseallow sequencing of a broader range of DNA templates. The novelfluorophore/linker combination, in the form of piperidine or piperazineaminoacid as the core molecule, disclosed in this invention, allows theconstruction of brighter ET dyes. The heterocyclic FRET cassettesdisclosed in this invention can be used to label nucleic acids,proteins, carbohydrates and other biological molecules of interest.

2. Description of Related Art

A large number of fluorescent dyes have been recently developed forlabeling and detecting components in biological samples. Generally,these fluorescent dyes must have high extinction coefficient and quantumyield so a low detection limit can be achieved.

One class of dyes which have been developed to give large and differentStokes shifts, based on the Foster Resonance Energy Transfer (FRET)mechanism and used in the simultaneous detection of differently labeledsamples in a mixture, are the ET (Energy Transfer) dyes. These ET dyesinclude a complex molecular structure consisting of a donor fluorophoreand an acceptor fluorophore as well as a labeling function to allowtheir conjugation to biomolecules of interests. Upon excitation of thedonor fluorophore, the energy absorbed by the donor is transferred bythe Forster Resonance Energy Transfer (FRET) mechanism to the acceptorfluorophore and causes it to fluoresce. Different acceptors can be usedwith a single donor to form a set of ET dyes so that when the set isexcited at one single donor frequency, various emissions can be observeddepending on the choice of the acceptors. Upon quantification of thesedifferent emissions, the components of a mixture can readily be resolvedwhen these dyes are conjugated to bio-molecules of interest. These ETdye sets constitute the backbone of current high throughput genesequencing methodology.

Previously, a variety of combinations of bi-fluorophore dyes have beendescribed. U.S. Pat. No. 5,688,648, entitled “Probes Labelled withEnergy Transfer Coupled Dyes” Mathies et.al., U.S. Pat. No. 5,728,528,entitled “Universal spacer/energy transfer dyes, and U.S. Pat. No.6,150,107, entitled “Methods of sequencing and detection using energytransfer labels with cyanine dyes as donor chromophores” which areincorporated herein by reference in its entirety, including anydrawings, discloses sets of fluorescent labels carrying pairs of donorand acceptor dye molecules wherein the labels can be attached to nucleicacid backbone for sequencing. The nucleic acid bases or the abasic sugarunits are used as spacers to separate the donor and acceptor dyes. Theoptimum distance for efficient energy transfer from the donor dye to theacceptor dye was found to be ˜6-10 bases. Included is a method foridentifying and detecting nucleic acids in a multi-nucleic acid mixtureby using different fluorescent labels, wherein the fluorescent moietiesare selected from families such as cyanine dyes and xanthenes. Thefluorescent labels comprise pairs of fluorophores where one fluorophoredonor has emission spectra, which overlaps the fluorophore acceptor'sabsorption so that there is energy transfer from the excited member tothe other member of the pair.

U.S. Pat. No. 6,008,373, entitled “Fluorescent labeling complexes withlarge stokes shift formed by coupling together cyanine and otherfluorochromes capable of resonance energy transfer” Waggoner et.al.,which is incorporated herein by reference in it's entirety, includingany drawings, discloses complexes comprising a first fluorochrome havingfirst absorption and emission spectra and a second fluorochrome havingsecond absorption and emission spectra. The linker groups between thefluorochromes are alkyl chains. The fluorescent nature of the dyesenables them to be of use in sequencing and nucleic acid detection.

U.S. Pat. No. 5,863,727, entitled “Energy transfer dyes with enhancedfluorescence” Lee et al., which is incorporated herein by reference inits entirety, discloses energy transfer dyes in which the donor andacceptor dyes are separated by a linker between the dyes. The preferredlinker between the dyes is 4-aminomethylbenzoic acid (Nucleic AcidsResearch, 1997, 25(14), 2816-2822). The energy transfer terminators DNAsequencing kit based on this linker is commercially available fromApplied Biosystems (Foster City, Calif.) and sold as Big Dye terminatorkit.

PCT application WO 00/13026 entitled “Energy Transfer Dyes” Kumar etal., which is incorporated herein by reference in its entirety,including any figures and drawings, discloses energy transfer dyes,their preparation, and their use as labels in biological systems. Thedyes are preferably in the form of cassettes, which enable theirattachment to a variety of biological materials. The donor dye, acceptordye and the dideoxynucleoside-5′-triphosphates are all attached to atrifunctional linker, which is based on aromatic aminoacids structure(Tetrahedron Letters, 2000, 41, 8867-8871). The energy transferterminator kit based on these structures is sold by AmershamBiosciences, Piscataway (N.J.) as DYEnamic ET terminator kit for DNAsequencing.

PCT application WO 01/19841 entitled “Charge-modified nucleic acidsterminators” Kumar et al., which is incorporated herein by reference inits entirety, including any figures and drawings, discloses single andenergy transfer dye labeled terminators with positive or negativecharge(s) incorporated in the linker arm. These terminators are usefulin generating DNA sequencing bands free of any ‘dye blobs’ which areformed by the degradation of dye labeleddideoxynucleoside-5′-triphosphates. The use of charge terminators allowsthese degradation products to move backward (positive chargeterminators) or move ahead of sequence information (negative chargeterminators, Finn et.al. Nucleic Acids Research, 2002, 30(13),2877-2885).

The currently available ET dye terminator sets, generally, suffer fromlow brightness. This low brightness is due to the inefficiencies in thetransfer of the energy absorbed by the donor to the acceptors andre-emission at the emission wavelength of the acceptor. Thisinefficiency arises because, the structural linkages used to join thedonor, the acceptors, and the nucleic acid bases together to form thedye terminators are less than optimal. Therefore, there remains a needfor additional improvements in energy transfer dye cassette constructionfor maximum brightness and attachment to biological molecules.

SUMMARY OF THE INVENTION

The current invention provides energy transfer dyes and labelednucleotides, which are brighter and are substrates for DNA polymerases.The energy transfer dyes use heterocyclic linker structures, such aspiperidine and piperazine as the core molecule, to attach the donor andacceptor dyes. For example, in case of piperidnyl-1,1-amino carboxylicacid, the donor dye is attached to the secondary nitrogen atom and theacceptor dyes are attached to the amino group. The carboxylic acidresidue of the molecule is used to attach the biological molecule ofinterest, such as a nucleoside, nucleotide, oligonucleotide or otherbiological molecule of interest. The attachment position of donor andacceptor dyes may also be switched.

The current invention also provides a set of four terminators derivedfrom the heterocyclic linkers of this invention. The terminator setinclude fluorescein (FAM) or rhodamine 110 (R110) as the donor dye andrhodamine 110 (R110), rhodamine 6G (R6G), tetramethylrhodamine (TAMRA)and rhodamine X (ROX) or cyanine dyes as the acceptor dyes. Thisterminator set is optimized for DNA sequencing. The labeled nucleotideterminators in the kit are brighter than the existing kits and giveuniform bands. The method of their preparation and use in DNA sequencingis also disclosed in the present invention.

Disclosed are compositions and methods of making the heterocyclic FRETdyes of this invention and their attachment to the biological moleculesof interest such as nucleosides, nucleotides (mono, di, ortriphosphates) or oligonucleotides.

The numbers and letters in bold represents the compound numbers given inschemes and in experimental sections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general scheme for the synthesis of piperidine linkerderived FRET cassettes and labeling dideoxynucleoside-5′-triphosphates

FIG. 2 is a plot of signal strength at 800 bases with differentconcentration of TAMRA labeled ddATP of different linker lengths

FIG. 3 shows single color electropherograms of FAM-Phe-TAMRA-11-ddATP(3F), FAM-Piperidine-TAMRA-11-ddATP (7), andFAM-Piperidine-TAMRA-18-ddATP (8) terminated amplicons.

FIG. 4 is a plot of signal strength at 800 bases with differentconcentration of ROX labeled ddCTP of different linker lengths

FIG. 5 shows single color electropherograms of FAM-Phe-ROX-11-ddATP(4F), FAM-Piperidine-ROX-11-ddATP (10), and FAM-Piperidine-ROX-18-ddATP(11) terminated amplicons.

DETAILED DESCRIPTION OF THE INVENTION

The efficiency of FRET depends on a number of factors. According toForster's theory (Joseph R. Lakowicz, “Principles of FluorescenceSpectroscopy” 2^(nd) Edition, Chapter 13, Kluwer Academic PlenumPublishers, New York, Boston, Durdrecht, London, Moscow 1999), theprimary factors are:

-   1) The overlap of the emission spectrum of the donor and the    absorption spectrum of the acceptor;-   2) The separation, in distance, between the donor and the acceptor;    and-   3) The spatial orientation between the dipoles of the donor and the    acceptor.

In practice, the situation is much more complicated. Specificinteractions between the donor and the acceptor, may in cases, lead toquenching with very little emission from the acceptor even when thedonor emission is completely absent. Furthermore, the extent to whichthe donor is quenched has very little bearing on the amount of energybeing transferred to the acceptor and, hence, the emission observed. Amathematical treatment to describe the practical ET process associatedwith these dye-terminators, has now been developed and is furtherdescribed below. In such a mathematical treatment, three experimentallymeasurable parameters are of paramount importance:

-   1) PQEQ (Percentage of quenching of the donor) which is defined as;    PQEQ=(1−Emission_(donor in the donor/acceptor pair)/Emission_(same amount of donor in the absence of the acceptor))×100%,-   2) PAEE (Percentage Acceptor Emission Efficiency):    PAEE=(Emission efficiency of the acceptor in the donor acceptor    pair)/(Emission efficiency of the acceptor without the donor)×100%,    and-   3) PET (Percentage Energy Transfer),    PET=Quantum yield of the donor×[(Emission efficiency of acceptor    when excited at the donor excitation wavelength)/(Emission    efficiency of the donor in the absence of the acceptor).-    PET, as defined, actually becomes the quantum yield of the    donor/acceptor pair when excited at the donor excitation wavelength    and the emission measured at the acceptor emission wavelength.

The above methodology can be extended to ET assemblies consisting of onedonor and more than two acceptors.

Furthermore, from these numbers, a flow diagram can be constructed toshow the photon flow throughout the ET process. As an example, thenumbers for the set of four ET dye-terminators (Kumar et.al. PCT WO00/13026; Nampalli et.al., Tetrahedron Letters 2000, 41, 8867) used incurrent DNA sequencing reactions are given in Table One below.

TABLE ONE (All measurements in 1 × TBE + 8 M urea buffer, 488 nmexcitation) Compound* PQEQ PAEE PET FAM-Phe-R110-11-ddGTP (1F) 92% Notmeasurable** 8% FAM-Phe-R6G-11-ddUTP (2F) 99% 47% 26%FAM-Phe-TAM-11ddATP (3F) 98% 48% 16% FAM-Phe-ROX-11ddCTP (4F) 99% 35%19% *The structures of these compounds are given in FIG. A. **Theemissions from FAM and R110 are not resolvable.

As an illustration, a photon flow diagram can be constructed using thecompound (4F) as an example.

The construction of the above diagram is relatively simple. Since PQEQfor the compound (4F) is equal to 99%, only one photon in 100 photonabsorbed by the donor (FAM) is re-emitted by the donor. We have PETequals to 0.19, it means that for the 100 photons absorbed by the donor,19 photons are emitted by the acceptor (ROX). Since the PAEE is 0.35,and assuming that the quantum yield of ROX in its free state is 1.0relative to FAM, we need the input of 19/0.35 or 54 photons to have 19photons emitted by ROX acceptor. It follows that the number of photonslost by the acceptor (ROX) in processes other than fluorescence must be35 (54−19). Then, from the conservation of photons, the number ofphotons lost in radiationless processes from the donor FAM should be 45(100−1−54).

During our extensive search for improved brightness of dye terminatorsets over those listed in Table One, we discovered a novel type of ETdyes based on piperidinyl-1, 1-amino carboxylic acid. The ring nitrogen(NH) of the piperidine nucleus is used to attach the donor dye(fluorescein). The amino group at the 1 position of the piperidine isused to attach the acceptor dyes (Rhodamine 110, Rhodamine 6G,Tetramethyl rhodamine, Rhodamine-X, Cy 5 etc.) and the biologicalmolecule such as nucleoside triphosphates, oligonucleotides, proteins isattached to the activated carboxylic acid. The ET dye cassettes andterminators derived from this architecture are brighter and the dideoxynucleotides are good substrate for DNA polymerasesThus this invention provides energy transfer dyes of the formula:

wherein

-   D is a donor dye selected from the group consisting of xanthine    dyes, rhodamine dyes, and cyanine dyes;-   L₁ is a functional group selected from the group consisting of H,    C₁-C₂₀ alkynylamine, alkynol, alkenamine, alkylamine, keto, and    thiol, through which D is covalently attached;-   R₁, R₂, R₃ and R₄ independently represent H, alkyl, halo, hydroxy,    thio, nitro, amino or alkylamino groups;-   L₂ is an amine selected from the group consisting of C₁-C₂₀    alkynylamine, alkynol, alkenamine, alkylamine, keto, and thiol,    through which A is covalently attached;-   X is an aldehyde, an acid, an acid chloride, an ester,    hydroxymethyl, CH₂O-mesylate, CH₂O-triflate, mercaptomethylene,    phosphoramidite or other reactive groups capable of forming a    covalent bond with amine, thiol, hydroxy, or haloacetyl containing    biological molecules; or-    X=L₃B wherein,-    L₃ is a functional group consisting of carboxylic acid,    N-hydroxy-succinimidyl ester, acid chloride, maleimide, hydrazide,    or sulfonyl chloride, capable of forming a covalent bond with    biological molecule, B-    B is a biological molecule selected from the groups consisting of    nucleosides, nucleoside-monophosphate, diphosphate, or    triphosphates, thiophosphates, alkenyl or alkynylamino substituted    dideoxynucleoside triphosphates, deoxynucleoside triphosphates,    nucleoside triphosphates, amino acids, proteins, or modified    oligonucleotides;-   A is an acceptor dye selected from the group consisting of xanthine    dyes, rhodamine dyes and cyanine dyes;-   wherein the positions of A and D are interchangeable.    In another embodiment the present invention provide the energy    transfer dye cassettes and the corresponding    dideoxynucleoside-5′-triphosphates of the following structures,    their preparation and use in DNA sequencing.    Wherein    Acceptor dye is selected from xanthine class of dyes, rhodamine    dyes, or cyanine dyes BASE is selected from cytosine, thymine,    uracil, adenine, guanine, hypoxanthine, 2,6-diaminopurine,    2-aminopurine, 7-deazapurines, 7-deaza-8-azapurines, and other    modified heterocyclic bases, and n is 0-3.    The acceptor dye and the donor dye may be attached interchangeably    at either of the ring nitrogen or the primary amino group of the    piperidine 1,1-amino carboxylic acid.    The ET dye cassettes for labeling can also be generated from the    other similar heterocyclic aminoacids, examples of which are shown    below.

EXAMPLES OF OTHER HETEROCYCLE DERIVED FRET SYSTEMS

In all the heterocycle derived FRET cassettes, the donor dyes (D)comprise the 5 and 6-regioisomers of the following: carboxyfluorescein(FAM), Cy3, rhodamine green (R110) and the acceptor dyes (A) comprisethe 5 and 6-regioisomers of the following: 5-carboxyrhodamine (R110),6-carboxyrhodamine, 5-carboxyrhodamine-6-G (R6G or REG),6-carboxyrhodamine-6-G,N,N,N′,N′-tetramethyl-5-carboxyrhodamine(TAMRA),N,N,N′,N′-tetramethyl-6-carboxyrhodamine, 5-carboxy-X-rhodamine(ROX), 6-carboxy-X-rhodamine,1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-carbocyanine(Cy3),1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)dibenzo-carbocyanine(Cy3.5),1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato)dibenzodicarbocyanine(Cy5), 1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)dibenzo-dicarbocyanine (Cy5.5),1-(ε-carboxypentyl)-1′-ethyl-3,3 and3′,3′-tetramethyl-5,5′-(1,3-disulphonato)tricarbocyanine (Cy7) orrelated dyes and wherein acceptor dye, A is capable of accepting energyfrom the donor dye, D.FAM, R110, REG, TAMRA, and ROX are trademarks of Applied Biosciences(Foster City, Calif.), Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 are trademarks ofAmersham Biosciences (Piscataway, N.J.)Additionally, the present invention includes the FRET cassettes (videsupra), which have a nitrogen atom in the ring systems, a 1,2- or1,3-related attachment to A and B. Note that these systems possess oneor two chiral centers, which influence the relative orientation of A, Band D, thereby influencing the energy transfer efficiencies (brightnessor quantum yield).

A number of single dye labeled terminators with different linkersbetween the dye and the dideoxynucleoside-5′-triphosphates weresynthesized and their brightness (PET) was measured by excitation at 488nm. The brightness of single dye labeled terminators was also comparedwith energy transfer dye labeled terminators of the present inventionand terminators previously disclosed. The synthesis of piperidinederived terminators was undertaken as shown in FIG. 1. Thus, as can beseen in Example 1, N-FMOC-piperidinyl-1,1-amino carboxylic acid (1) wasreacted with the protected fluorescein-5-carboxylic acid chloride (3) togive the fluorescein labeled piperidinyl-1,1-amino carboxylic acid (4).The acceptor dyes (in the form of NHS esters) were attached to the aminogroup after deprotection with piperidine. Finally, the activation ofacid to the corresponding active ester followed by reaction withappropriately linked propargylamino-ddNTPs provided the piperidinelinker derived energy transfer dye terminators. Alternatively,N-t-Boc-piperidinyl-1,1-carboxylic acid methyl ester can be used tosynthesize single and ET dye cassettes of the present invention.

The energy transfer efficiency (PET) was measured for all the single dyelabeled dideoxynucteoside-5′-triphosphates (terminators) and the energytransfer terminators synthesized in this invention. All the dyeterminators were excited at 488 nm and emission was measured at theirrespective emission wavelengths. The results are provided in Table 2,below.

TABLE 2 (All measurements in 1 × TBE + 8 M urea buffer, 488 nmexcitation) Dye terminator PET FAM-18-ddGTP (I)  38* R110-18-ddGTP (II) 28* R6G-11-ddUTP (III)  28* TAMRA-11-ddATP (IV)  1* ROX-11-ddCTP (V) 1* FAM-piperidine-TAMRA-11-ddATP (7) 21 FAM-piperidine-TAMRA-18-ddATP(8) 41 FAM-piperidine-ROX-11-ddCTP (10) 40 FAM-piperidine-ROX-18-ddCTP(11) 60 *For single dye terminators, PET equivalent = quantum yield ofthe Single dye-terminator × (extinction coefficient at 488nm)/(extinction coefficient at absorption maximum). **Emission from theR110 can not be resolved from that of FAM.The molecular structures of the dye terminators listed in TABLE 2 aregiven below.

-   -   I: Dye=FAM, n=2, ddNTP=ddGTP    -   II: Dye=R110, n=2, ddNTP=ddGTP    -   III: Dye=R6G, n=1, ddNTP=ddUTP    -   IV: Dye=TAMRA, n=1, ddNTP=ddATP    -   V: Dye=ROX, n=1, ddNTP=ddCTP        Structures of Single Dye Labeled        dideoxynucleoside-5′-triphosphates (Terminators)        The single dye labeled terminators and energy transfer dyes        labeled terminators of this invention (described above) were        tested in DNA sequencing reactions using thermostable DNA        polymerase. The utility of individual dye labeled terminator was        ascertained based on the overall sequence quality, brightness,        and uniformity of the bands. Based on these criterions, a new        set of dye-terminators was constructed. The PET of terminators        of this new set is given below.

PET Dye-terminator or equivalent DYEnamic ET set FAM-18-ddGTP (IX), 38 8 (1F) R6G-11-ddUTP (XI), 28 26 (2F) FAM-piperidine-TAM-18-ddATP (8),41 16 (3F) FAM-piperidine-ROX-18-ddCTP (11) 60 19 (4F)

The invention also includes a reagent and a method for making thereagent including incubating the fluorescent water-soluble labelingcomplex described above with a carrier material. The complex or thecarrier material having a functional group that will react with areactive group of the other of the complex or the carrier to form acovalent bond between them. The carrier material can be selected fromthe group consisting of polymer particles, glass beads, cells,antibodies, antigens, proteins, enzymes, and nucleotides derivatized tocontain one of an amino, sulfhydryl, carbonyl, carboxyl, or hydroxylgroups. Alternatively, the carrier material may contain the reactivegroups and the fluorescent labeling complex of the invention may containany of the aforementioned functional groups that will react with thereactive group to form covalent bonds.

In an alternative embodiment, the fluorescent complexes of the inventionneed not have a reactive group when used to non-covalently bind toanother compound. For example, the complex may be dissolved, then mixedin an organic solvent with a polymer particle, such as polystyrene andthen stirred by emulsion polymerization. The solvent is evaporated andthe fluorescent dye complex is absorbed into the polystyrene particles.

The invention is further described by reference to the followingexamples. These examples are provided for illustration purposes only andshould not be construed as limiting the appended claims and the scope ofthe invention. The current invention should encompass any and allvariations that become evident from the teachings provided herein.Numbers that appear in bold below refer to the various numberedcompounds in the synthesis.

Example 1 Synthesis of Piperidine Linker Derived Terminators(FAM-Piperidine-ROX-X-ddNTPs)

4-N-(dipivaloylfluorescein-5-carbonyl)-N-FMOC-piperidinyl-1,1-aminocarboxylic acid (4)

N-FMOC-piperidinyl-1,1-amino carboxylic acid 1 (0.4 g, 1.0 mmol) wasdried by coevaporation with anhydrous pyridine (10 ml). The driedsubstrate was dissolved in a mixture of methylene chloride (5 ml) andpyridine (5 ml). The reaction flask was cooled in an ice bath and asolution of dipivaloyl-5-carboxyfluorescein acid chloride 3 (1.25 mmol,prepared by treating dipivaloyl-5-carboxyfluorescein 2 with oxalylchloride in methylene chloride in the presence of DMF) in methylenechloride (10 ml) was added. The reaction mixture was stirred at 0-5° C.for 2 h and allowed to warm to room temperature. The reaction wascontinued overnight and quenched by the addition of water (0.5 ml). Thereaction mixture was diluted with chloroform (100 ml) and washed withwater (50 ml). Organic layer was dried (sodium sulfate) and evaporated.The residue was coevaporated with toluene and purified by silica gelcolumn chromatography using a gradient of 0-5% methanol in methylenechloride as the eluent.

4-N-(fluorescein-5-carbonyl)piperidinyl-1,1-amino carboxylic acid (5)

To a solution of compound 4 (150 mg) in THF (15 ml) piperidine (5 ml)was added and the mixture was stirred at room temperature for 18 h. Thereaction mixture was evaporated to dryness and the residue was purifiedby column chromatography using a gradient of 0-100% methanol inmethylene chloride as the eluent.

4-N-(fluorescein-5-carbonyl)-N-ROX-piperidinyl-1,1-amino carboxylic acid(6)

A mixture of the compound 5 (20 mg) and ROX-NHS ester was dried bycoevaporation with dry DMF (15 ml). Then it was dissolved in anhydrousDMSO (5 ml) to which N,N-diisopropylethyl amine (0.5 ml) was added andthe reaction mixture was stirred at room temperature for 6.5 h. Thereaction mixture was loaded on a silica gel column, which was packed inmethylene chloride. The product was eluted utilizing 0-100% methanol inmethylene chloride followed by 1%-trifluoroacetic acid in methanol. Thefractions containing the product were evaporated and the residue wascoevaporated with toluene. The residue was further purified on aQ-Sepharose column. Eluent: 0.1 N triethylammonium bicarbonatecontaining 40% acetonitrile to 1N triethylammonium bicarbonatecontaining 40% acetonitrile. The fractions containing the product werecollected and evaporated to give 6.

5-FAM-piperidine-ROX-11-ddCTP (10).

Compound 6 (10 mg, 0.01 mmol) was dried by coevaporation with dry DMF (8ml). Then it was dissolved in dry DMF (3 ml) to which a solution ofdisuccinimidyl carbonate (18 mg) in DMF (1 ml) was added. The reactionflask was cooled to −60 ° C. and a solution of DMAP (9 mg) in DMF (1 ml)was added dropwise. After 10 min the reaction mixture was allowed towarm to −30° C. and a solution of 11-ddCTP (0.3 mmol) in pH 9.5NaHCO₃/Na₂CO₃ buffer (8 ml) was added. The reaction mixture was allowedto warm to room temperature and the reaction continued for 3 h. Thereaction mixture was directly loaded on a silica gel column, which waspacked in 50% methanol-chloroform. The column was washed with methanoland the product was eluted using a mixture of isopropanol, ammoniumhydroxide and water (6:3:1). The fractions containing the product werecollected and evaporated to a small volume, filtered through 0.4 μfilter and loaded on a Q-sepharose column. The product was eluted with agradient of 0.1N triethylammonium bicarbonate containing 40%acetonitrile to 1 N triethylammonium bicarbonate containing 40%acetonitrile. The appropriate fractions containing the pure product werecollected and evaporated. The residue was coevaporated with methanol(5×30 ml) to afford compound 10.

5-FAM-Piperidinyl-ROX-18-ddCTP (11)

FAM-piperidinyl-ROX, the ET cassette 6 (4 mg 0.004 mmol) was dissolvedin anhydrous DMF(2 ml). A total of 8 mg (0.03 mmol, 8 eq) DSC was added.The mixture was stirred and cooled to −60 ° C., at this temperature, ananhydrous DMF solution (1 mL) of DSC (2.4 mg was added dropwise. After15 minutes TLC indicated complete conversion to the NHS ester. Thereaction mixture was warmed up to −30° C. and a buffer (0.1 MNa₂CO₃—NaHCO₃; pH 8.5) solution of 18-ddCTP (1 eq) was added. Then, thereaction mixture was allowed to warm up to room temperature and stirredfor another 3 hours. The desired product 11 was purified on aQ-Sepharose column as described for 10.

Example 2 Synthesis of FAM-Piperidine-TAMRA-X-ddNTPs

4-N-(fluorescein-5-carbonyl)-N⁴-TAMRA-piperidinyl-1,1-amino carboxylicacid (9)

Compound 5 (24 mg, 0.048 mmol) was dissolved in anhydrous DMSO (5 mL)and to the stirred solution at room temperature was added DIPEA (0.1 mL,0.58 mmol, 12 eq) followed by TAMRA-NHS ester (30 mg, 0.57 mmol, 1.2eq). The reaction mixture was stirred overnight and the desired product9 (60%) was isolated using a Q-Sepharose column as described forcompound 6.

5-FAM-Piperidinyl-TAMRA-11-ddATP (7) and5-FAM-Piperidinyl-TAMRA-18-ddATP (8)

Compounds 7 and 8 were synthesized from 9 on a 3.8 μmole scale using 8equivalents of DSC, 5 equivalents of DMAP and 1 equivalent of 11- or18-ddATP following similar reaction conditions described for 10.

Example 3 Synthesis of FAM-Piperidine-R110-X-ddNTP andFAM-Piperidine-R6G-X-ddNTP 5-FAM-Piperidinyl-R110-11-ddGTP (15)

Terminator 15 was synthesized and purified from 5 via 14 following theprocedure similar to that of 7.

Example 4 Synthesis of a single dye (R110 as the donor dye) cassettederived from Piperidine Piperidinyl-R110 Cassette Synthesis (19)

TFA-5-R110 acid 17 (0.2 g, 0.35 mmol) was dried by coevaporation withdry DMF (10 ml). The dry material was dissolved in anhydrous THF (6 ml).The reaction flask was cooled in ice bath and a drop of DMF followed byoxalyl chloride (0.25 ml, 0.5 mmol) was added in over 5 min time. Thereaction was continued for 15 min in ice bath and then allowed to warmto room temperature. After 2 h at room temperature evaporated todryness, coevaporated with dry methylene chloride (10 ml) and driedunder high vacuum for 1.5 h to provide 18.FMOC-piperidine 1 derivative (0.14 g) was dried by coevaporation withdry pyridine (10 ml). Then it was dissolved in pyridine (3 ml) to whicha solution of acid chloride (18) in a mixture of methylene chloride (5ml) and anhydrous acetonitrile (1 ml) was added dropwise at 0° C. Thereaction mixture was stirred at 0° C. for 3 h and allowed to warm toroom temperature. After stirring the reaction mixture at roomtemperature for 15 h, the reaction was quenched by the addition of water(1 ml). The reaction mixture was diluted with methylene chloride andwashed with water. Organic layer was dried (sodium sulfate), evaporatedand coevaporated with toluene. Finally the product was purified bysilica gel column chromatography using 0-8% methanol-methylene chlorideas the eluent. The appropriate fractions containing the desired productwere pooled and evaporated to give 19. Compound 19 can be converted to21 via 20 for the generation of R110-Piperidine-Rhodamine ET cassetteand its corresponding terminators.The efficiency of FRET in terms of percent energy transfer (PET) wasmeasured between the fluorescein and rhodamine dyes of the heterocyclicFRET-cassettes and terminators on a fluorimeter (Photon TechnologyInternational) in 1×TBE, 8 M Urea and compared with the single-dyelabelled-terminators. It is clear from the plotted bar graph that theFRET cassettes and terminators of the present invention showed increasedPET. The longer spacer terminator (11) displayed 55 times enhanced PETthan that of ROX-11-ddCTP (single-dye labelled-terminator) and 3.2 timesthan that of commercial 4F, while the shorter spacer terminator (10)showed 2.1 times enhanced PET over 4F. The next terminator,FAM-Piepridine-TMR-18-ddATP (8), whose fluorescence enhancement is alsodesirable as it is far removed in the spectrum for overlapping with itsabsorption, showed 41 times increased PET over the single-dyelabelled-terminator and 2.5 times over the commercial 4F.

Synthesis of FAM-Piperidine-Cy5-X-ddNTPs

The synthesis of energy transfer terminators with Cy5 as an acceptor dyecan be carried out exactly the same way as given in example 1 and 2. Cy5NHS ester is used in place of rhodamine-NHS ester.

Example 5 Sequencing DNA Using Single and Energy Transfer Dye LabeledDideoxynucleoside Triphosphates

A sequence of M13mp 18 template DNA was generated using standard “−40”primer. The reaction mixture (20 μl) contained 200 μM each of dATP,dCTP, and dTTP, 1000 μM dITP, 160 nM FAM-18-ddGTP, 125 nM R6G-11-ddUTP,95 nM FAM-Piperidine-TMR-18-ddATP, 60 nM FAM-Piperidine-ROX-18-ddCTP, 2pmol −40 primer, 200 ng M13mp 18 DNA, 20 units of Thermo Sequenase orother mutated DNA polymerase (Amersham Biosciences), 0.0008 unitsThermoplasma acidophilum inorganic pyrophosphatase, 50 mM Tris-HCl pH8.5, 35 mM KCl and 5 mM MgCl₂.

The reaction mixture was incubated in a thermal cycler for 25 cycles of95° C., 20 Sec; 50° C., 30 Sec., and 60° C., 120 Sec. After cycling, thereaction products were precipitated with ethanol using standardprocedures, washed and resuspended in formamide loading buffer. Thesample was loaded on an Applied Biosystems model 377 instrument orMegaBACE 1000 (Amersham Biosciences) and results were analyzed usingstandard software methods.

Those skilled in the art having the benefit of the teachings of thepresent invention as set forth above, can effect numerous modificationsthereto. These modifications are to be construed as being encompassedwithin the scope of the present invention as set forth in the appendedclaims.

1. An energy transfer dye labeled cassette of the formula:

wherein D is a donor dye selected from the group consisting of xanthinedyes, rhodamine dyes, and cyanine dyes; L₁ is a functional groupselected from the group consisting of C₁-C₂₀alkynyl-amine, alkynol,alkenamine, alkylamine, keto, and thiol, through which D is covalentlyattached; R₁, R₂, R₃ and R₄ independently represent H, alkyl, halo,hydroxy, thio, nitro, amino or alkylamino groups; L₂ is an amineselected from the group consisting of C₁-C₂alkynylamine, alkynol,alkenamine, alkylamine, keto, and thiol, through which A is covalentlyattached; X is an aldehyde, an acid, an acid chloride, an ester,hydroxymethyl, CH₂O-mesylate, CH₂O-triflate, mercaptomethylene,phosphoramidite or other reactive groups capable of forming a covalentbond with amine, thiol, hydroxy, or haloacetyl containing biologicalmolecules; A is an acceptor dye selected from the group consisting ofxanthine dyes, rhodamine dyes and cyanine dyes; and wherein thepositions of A and D are interchangeable.
 2. An energy transfer dyelabeled compound of the formula:

wherein D, L₁, L₂, R₁, R₂, R₃, R₄, and A are defined as in claim 1; L₃is a functional group consisting of carboxylic acid,N-hydroxy-succinimidyl ester, acid chloride, maleimide, hydrazide, orsulfonyl chloride, capable of forming a covalent bond with biologicalmolecule, B; B is a biological molecule selected from the groupsconsisting of nucleosides, nucleoside-monophosphate, diphosphate, ortriphosphates, thiophosphates, alkenyl or alkynylamino substituteddideoxynucleoside triphosphates, deoxynucleoside triphosphates,nucleoside triphosphates, amino acids, proteins, or oligonucleotides;and wherein the positions of A and D are interchangeable.
 3. An energytransfer dye and dye labeled compound of the formula:

wherein D, L₁, L₂, A, L₃, and B are defined as in claim 2; Z is H,alkylene, alkyl, aryl or combination there of; and wherein the positionsof A and D are interchangeable.
 4. The energy transfer dye labeledcompound of claim 2 or 3 wherein B is alkenyl or alkynylaminosubstituted nucleoside triphosphate or an oligonucleotide.
 5. The energytransfer dye labeled compound of claim 2 or 3 wherein B is a alkenyl oralkynylamino substituted dideoxynucleoside-5′-triphosphate.
 6. Theenergy transfer dye labeled compound of claim 2 or 3, wherein L₃ isattached to the C-5 position of pyrimidines and at the C-7 position of7-deazapurines through a alkynyl, alkenyl or saturated side chain. 7.The energy transfer dye labeled compound of any of claims 1, 2 or 3,wherein said D and A are selected from the group consisting of5-carboxyrhodamine, 6-carboxyrhodamine, 5-carboxyrhodamine-6-G,6-carboxyrhodamine-6-G, N,N,N′,N′-tetramethyl-5-carboxyrhodamine,N,N,N′,N′-tetramethyl-6-carboxyrhodamine, 5-carboxy-X-rhodamine,6-carboxy-X-rhodamine,1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-carbocyanine,1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)dibenzo-carbocyanine,1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato)dibenzo-dicarbocyanine,1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)dibenzo-dicarbocyanine,1-(ε-carboxypentyl)-1′-ethyl-3,3 and 3′,3′-tetramethyl-5,5′-(1,3-disulphonato)tricarbocyanine or related dyes and whereinacceptor dye, A is capable of accepting energy from the donor dye, D. 8.A method for determining the nucleotide base sequence of a DNA moleculecomprising: incubating a DNA molecule annealed with a primer moleculeable to hybridize said DNA molecule in a vessel containing athermostable DNA polymerase, a compound according to claim 2 or 3; andseparating DNA products of the incubating reaction according to sizewhereby at least a part of the nucleotide base sequence of said DNAmolecule can be determined.
 9. A kit for DNA analysis comprising acompound of claim 2 or 3 and a DNA polymerase.
 10. A compound of theformula

wherein A is an acceptor dye according to claim 1 above, and BASE isselected from the group consisting of cytosine, thymine, uracil,adenine, guanine, hypoxanthine, xanthine, 2,6-diaminopurine,2-aminopurine, 7-deazapurines, 7-deaza-8-azapurines, and homologsthereof, and n is 0-3.
 11. A kit for DNA sequencing comprising acompound of claim
 10. 12. A kit according to claim 11, furthercomprising a DNA polymerase.
 13. A deoxyribonucleic acid sequencecomprising one or more compounds of claim 10 in monophosphate form. 14.Method of determining the nucleotide base sequence of a DNA moleculecomprising: a) incubating a DNA molecule annealed with a primer moleculeable to hybridize to said DNA molecule in a vessel containing athermostable DNA polymerase, a compound of claim 10; and b) separatingDNA products of the incubating reaction according to size whereby atleast a part of the nucleotide base sequence of said DNA molecule can bedetermined.
 15. A compound according to claim 10 of the formula

wherein BASE is selected from the group consisting of adenine,7-deaza-adenine, guanine, 7-deaza-guanine, uracil, cytosine,hypoxanthine and 7-deaza-hypoxanthine.
 16. A compound according to claim10 of the formula

wherein BASE is selected from the group consisting of adenine,7-deaza-adenine, guanine, 7-deaza-guanine, uracil, cytosine,hypoxanthine and 7-deaza-hypoxanthine.
 17. A compound according to claim10 of the formula

wherein BASE is selected from the group consisting of adenine,7-deaza-adenine, guanine, 7-deaza-guanine, uracil, cytosine,hypoxanthine and 7-deaza-hypoxanthine.
 18. A compound according to claim10 of the formula

wherein BASE is selected from the group consisting of adenine,7-deaza-adenine, guanine, 7-deaza-guanine, uracil, cytosine,hypoxanthine and 7-deaza-hypoxanthine.
 19. A compound according to claim10 of the formula

wherein BASE is selected from the group consisting of adenine,7-deaza-adenine, guanine, 7-deaza-guanine, uracil, cytosine,hypoxanthine and 7-deaza-hypoxanthine.